Transducin, consisting of alpha (Gtalpha) and beta/gamma (Gtbeta/gamma) subunits, is the most abundant heterotrimeric guanine nucleotide binding (G) protein in vertebrate retina. Gtalpha, on interaction with receptor and Gtbeta/gamma, releases GDP and binds GTP. Gtalpha in the GTP-bound activated state dissociates from Gtbeta/gamma and stimulated the effector cGMP phosphodiesterase. The amino terminus of Gtalpha is critical to its overall function, as this area interacts with Gtbeta/gamma, an interaction that is facilitated by the presence of a myristoyl or similar acyl modification of the amino terminal glycine. Two monoclonal antibodies, LAS1 and LAS2, made against purified bovine Gtalpha were mapped with high precision to the amino terminus of Gtalpha by proteolytic modification and recombinant mutant proteins. Trypsin, which removes a-2 kDa fragment from the amino terminus of the 39-kDa Gtalpha, generated a 37-kDa product that failed to react with either of the antibodies on immunoblots. After Arginine-C proteolytic cleavage, reactive products were found at 34 kDa, 23 kDa and 15 kDa, all of which were unable to be sequenced by Edman degradation consistent with these fragments containing a blocked amino terminus. The amino terminal modification with an acyl group is required for antibody recognition, as synthesis of recombinant Gtalpha in E. coli without myristoylation resulted in no immunoreactive protein. When coexpressed in E. coli with N-myristoyltransferase, the myristoylated protein reacted with both antibodies. Recombinant Goalpha or a Gtalpha/Goalpha chimeric protein consisting of amino acids 1 to 9 of Gtalpha and the remainder Goalpha did not react with the antibodies whether or not myristoylated. A myristoylated chimera of Gtalpha amino acids 1 to 17 fused with Goalpha reacted with both LAS antibodies. This confirmed that amino acids 10 to 17 of Gtalpha along with the fatty acyl group were necessary for reactivity, consistent with the notion that the fatty acyl group folds back upon the amino terminus of Gtalpha to form the epitope. Although by all physical mapping studies the epitopes of these antibodies appear to be identical, they differ functionally in their ability to inhibit the Gtalpha-dependent pertussis toxin-catalyzed ADP-ribosylation of Gtalpha, LAS2, but not LAS1, inhibited this reaction.